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Analysis of Lambda DNA with Restriction Enzymes Module III
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Analysis of Lambda DNA with Restriction Enzymes (Module III)
-black = marker + HindIII
-green = EcoRI
-pink = PstI
Purpose
--> shorther DNA segments travel farther in the gel
= anti-proportional relationship
Procedure
5
3
4
1
2
- EcoRI, HindIII, and PstI enyzmes
- Creation of lambda DNA fragments
- Gel electrophoresis:size of fragments
- What are palindromes?
Electrophoresis chamber was set up
Heated on heating block (65°C for 5 min)+ iced
Waterbath (37°C for 30 min)
2ul loading dye added to each tube
Prepare test tubes (4 ul DNA, 5ul buffer, 1 ul enzymes) + mix
Lambda
- The lambda DNA was not digested and stayed the same resulting in one big clump at the top of the gel
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9
6
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Background
PstI
Gel stained and washed (2x)
Examine results
Samples electrophoresed (300V for 10-15 min)
10 ul of each sample + marker added to seperate wells
HindIII
- PstI digested resulting into 8 bands in the electrophoresis gel
- What are Restriction Enymes ?
- Lambda DNA
- Fragment DNA
- Restriction sites
- EcoRI, HindIII, PstI
- Gel Electrophoresis
HindIII digested resulting in 6 bands in the electrophoresis gel
Click to edit text
What is our
EcorI
Conclusion?
Summary
EcorI digested resulting into 6 bands in the electrophoresis gel
--> not ten bands = hypothesis not supported
Analysis of Lambda DNA
By Noemi Morgan, Jaelyn Browne,
Katharina Blume, Seth Wright
- Different restriction enzymes cut the DNA at specific restriction sites (palindromic sequences)
-> segments of different sizes (basepairs)
Strands read the same in 5´to 3´ direction
HindIII
Materials
Minicentrifuge
- 1 starch 1% agarose gel (pre-casted)
- 1 electrophoresis chamber
- HindIII, EcoRI and PstI
- Lambda DNA
- Restriction Buffer
- Heating block
- Micropipettes
- Empty beaker for disposing pipette tips
- 6 microcentrifuge tubes
- 1 microcentrifuge tube holder
- Loading dye
- DNA marker
- 1 Staining tray
- Ziplock bag
What are the
real life applications?
Results: Theoretical Data
P = PstI restriction digest of lambda DNA
E = EcoRI restriction digest of lambda DNA
H = HindIII restriction digest of lambda DNA
M= DNA marker
L = Uncut lambda DNA
--> count and meassure the distance of the bands to the well
Hypothesis
--> compare the bands to the DNA marker with known basepair lengths
Lambda digested by EcoRI generates 10 bands in the gel electrophoresis.
Results: Experimental
- DNA marker does not show properly
--> can not compare samples
- no/only few bands for the rest of the samples
Results
1.
DNA fingerprinting.
2.
Methylation analysis
Genetic engineering
3.
4.
The Medical field